Method for producing Leu13 !motilin

ABSTRACT

A novel motilin analog peptide containing leucine instead of the usual 13th amino acid methionine, as in the naturally occurring motilin, is produced by gene recombination techniques in which several or identical genes, each coding for the novel peptide, are joined in series into a vector. The resultant recombinant DNA is introduced into Escherichia coli and the resultant transformant is cultivated. The resulting polymeric peptide can be cleaved to give the desired peptide.

This is a division of application Ser. No. 08/387,566, filed Feb. 13,1995 which is a continuation of Ser. No. 08/094,915, filed Jul. 22,1993, now U.S. Pat. No. 5,420,431 which is a continuation-in-part ofSer. No. 07/781,655 filed Oct. 25, 1991 abandoned which is acontinuation of Ser. No. 07/602,388 filed Oct. 24, 1990 abandoned whichis a continuation of Ser. No. 07/094,886 filed Sep. 10, 1987 abandoned.

FIELD OF THE INVENTION

This invention relates to a novel peptide which is a motilin analogcontaining leucine (Leu) in lieu of the 13th amino acid methionine (Met)of motilin, to DNAs coding for said peptide, to recombinant DNAscontaining said DNAs, and to methods of producing these.

The novel peptide (hereinafter referred to as Leu¹³ !motilin) accordingto the invention is comparable in activity to naturally occurringmotilin, hence can be expected to be useful as a drug.

BACKGROUND OF THE INVENTION

Motilin is a physiologically active peptide occurring in the mammalianblood and is known to be capable of activating peristalsis of theintestine (W. Y. Chey and K. Y. Lee, Clinics in Gastroenterology, 3, 645(1980)). Laparotomized patients show decreased motilin concentrations inthe blood, and is known that the return of postoperative blood motilinconcentrations to a normal level is correlated with the restoration ofperistaltic movement of the intenstine in such patients and thatpost-operative administration of motilin results in activatedperistalsis of the intestine.

Natural motilin can be obtained by extraction from animal organs but ininsufficient quantities. Therefore, motilin in current use is mostly aproduct of chemical peptide synthesis. However, this chemical product isnecessarily expensive since motilin is a relatively long chain peptidecomposed of 22 amino acid residues. Accordingly, it is desired that asubstance having motilin activity be supplied at low cost and in largequantities.

The 13th amino acid of motilin is Met, which is readily oxidizable.Oxidation of Met to the sulfoxide form results in decreased motilinactivity (M. Fujino et al., Chem. Pharm. Bull., 26, 101 (1978)).

SUMMARY OF THE INVENTION

AS a result of their investigations made in an attempt to find a methodof preventing the loss of motilin activity, the present inventors foundthat a peptide resulting from replacement in motilin of the 13th aminoacid Met with Leu, namely Leu¹³ !motilin, is comparable in activity tomotilin but does not suffer the loss of activity caused by oxidation.

Furthermore, as a result of their investigations made in an attempt tofind a method of supplying Leu¹³ !motilin at low cost and in largequantities, the inventors found that Leu¹³ !motilin can be suppliedutilizing gene recombination techniques.

It is said that large scale production of small-molecule peptides usinggenetic engineering techniques is generally difficult. This issupposedly because the small-molecule peptides produced in host cells,for example, microbial cells, are readily decomposed under the action ofenzymes in those cells. For preventing such decomposition, a method hasbeen reported which comprises producing the desired peptide in the formof a high-molecular-weight fused protein resulting from fusion of thedesired peptide with another protein, then decomposing, eitherenzymatically or chemically, the fused protein to yield the desiredpeptide (T. Mikuni et al., Seikagaku, 57, 854 (1985) and T. Saito etal., Biochem., 102, 111 (1987)).

Another method has been proposed which comprises joining the gene codingfor the desired peptide in series, producing the polymeric peptide, thendecomposing enzymatically or chemically the polymeric peptide to obtainthe desired monomeric peptide.

It has been reported that human proinsulin can be present in Escherichiacoli more stably in the polymeric form than in the monomeric one (S. -H.Shen, Proc. Natl. Acad. Sci. USA, 81, 4627 (1947)). However, in thisprior art, an attempt of conversion of the polymeric peptide to thedesired monomeric peptide were not made.

The above-described method was applied to the production of Substance P(T. Kempe et al., Gene, 39, 239 (1985)) and growth hormone releasingfactor (T. Kempe et al., Biotechnology, 4, 565 (1986)). In these cases,the monomeric peptides which are obtained by decomposing the polymericpeptide produced are different in the structure from the desirednaturally occurring peptide.

As a matter of course, however, the yield of the desired peptideobtainable by the above method is low since the fused protein soproduced contains only a small percentage of the desired peptide.

The present inventors found that the novel peptide according to theinvention can be produced in a polymeric from by inserting a pluralityof different or identical genes each coding for said peptide joined inseries into a vector, introducing the resultant recombinant DNA intoEschericia coli and cultivating the resultant transformant, thencleaving the polymeric peptide to give the desired peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings,

FIG. 1 shows a construction scheme for the plasmid pMTA1;

FIG. 2 shows a construction scheme for the plasmid pMTA2, wherein Bg,Bam and P indicate the sites of recognition by BglII, BamHI and PstI,respectively, and MT indicates a Leu¹³ !motilin gene (hereinafter thesame designations shall apply);

FIG. 3 shows a construction scheme for the plasmid pMTA4;

FIG. 4 shows a construction scheme for the plasmid pMTK4, wherein SGHindicates the salmon growth hormone gene (hereinafter the samedesignation shall apply);

FIG. 5 shows a construction scheme for the plasmid pMTL4, wherein Tippindicates the lipoprotein terminator and Trp indicates the tryptophanpromoter;

FIG. 6 shows a construction scheme for the plasmid pMTN4;

FIG. 7 shows a construction scheme for the plasmid pMTM4;

FIG. 8 shows a construction scheme for the plasmid pMTO4;

FIG. 9 shows a construction scheme for the plasmid pMTOI4;

FIG. 10 shows a construction scheme for the plasmid pMTOII4;

FIG. 11 shows a construction scheme for the plasmid pMTOIII4;

FIG. 12 shows a construction scheme for the plasmid pTrS20;

FIG. 13 shows a construction scheme for the plasmid pGHD7;

FIG. 14 shows a construction scheme for the plasmid pArg4;

FIG. 15 shows a construction scheme for the plasmid pGEL10;

FIG. 16 shows a construction scheme for the plasmid psGHIM1; and

FIG. 17 shows the intestine-contracting activity of Met¹³ !motilin andthat of Leu¹³ !motilin, wherein ∘ is for Leu¹³ !motilin and  for Met¹³!motilin.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus provides a novel peptide ( Leu¹³ !motilin) having theamino acid sequence defined by the following formula: ##STR1## whereinthe symbols represent the respective amino acid residues as follows:Phe, phenylalanine, Val, valine; Pro, proline; Ile, isoleucine; Thr,threonine; Tyr, tyrosine; Gly, glycine; Glu, glutamic acid; Leu,leucine; Gln, glutamine; Arg, arginine; Lys, lysine; and Ash, asparagine(hereinafter the same designations shall apply throughout thespecification and appendel claims).

Leu¹³ !motilin can be synthesized by the solid phase method of peptidesynthesis (G. Barang et al., "The Peptide: Analysis, Synthesis, Biology"(edited by E. Gross and J. Meienhofen), vol. 2, p.1, Academic Press(1982)) using an automatic peptide synthesizer (e.g. Beckman model990B).

Leu¹³ !motilin can also be produced by making the most use of geneticengineering techniques as follows:

Step 1 (cf. FIG. 1)

Firstly, DNAs defined by the formulae given below (Formulae 3-6) arechemically synthesized:

    5' CGATCAGATCTTCATGTTCGTTCCGATTTTCACTTACGGTGAACTGCAAC 3' (Formula 3)SEQ ID NO:2

    5' AGTTCACCGTAAGTGAAAATCGGAACGAACATGAAGATCTGAT 3' (Formula 4)SEQ ID NO:3

    5' GTCTGCAAGAGAAAGAACGTAACAAAGGTCAGCGGATCCTGTAAGAGCT 3' (Formula 5)SEQ ID NO:4

    5' CTTACAGGATCCGCTGACCTTTGTTACGTTCTTTCTCTTGCAGACGTTGC 3' (Formula 6)SEQ ID NO:5

In the above formulae and hereinafter, A, T, G and C represent the basesadenine, thymine, guanine and cytosine in the nucleotides, respectively.

These DNAs are synthesized in the manner of solid phase synthesis by thephosphoramidide method using an automated DNA synthesizer.

The double-stranded DNA fragment formed from the DNA of Formula 3(hereinafter, DNA 3) and the DNA of Formula 4 (hereinafter, DNA 4) andthe one formed from the DNA of Formula 5 (hereinafter, DNA 5) and theDNA of Formula 6 (hereinafter, DNA 6) are joined together using ligase.Thus is constructed a double-stranded DNA of the formula given below(hereinafter referred to as "gene 2"). ##STR2## wherein the lead linesdirected to the symbols ClaI, BglII, BamHI and SacI indicate the sitesof cleavage by the respective restriction enzymes represented by saidsymbols.

This gene 2 has a base sequence coding for a peptide composed of 30amino acids with aspartic acid (Asp)-glutamine (Gln)-isoleucine(Ile)-phenylalanine (Phe)-methionine (Met) being bound to the aminoterminus of Leu¹³ !motilin and arginine (Arg)-isoleucine (Ile)-leucine(Leu) being bound to the carboxyl terminus. The DNA segments coding forthe amino acids bound to the amino terminus and carboxyl terminus ofLeu¹³ !motilin contain sites recognizable by the restriction enzymesBglII and BamHI and these sites are of use in constructing genes codingfor Leu¹³ !motilin polymers. Gene 2 has been designed such that twoneighboring Leu¹³ !motilin monomers in the polymers produced on thebasis of these genes are connected to each other via a peptide (spacerpeptide) composed of 4 amino acids, Arg-Ile-Phe-Met. This spacer peptidecan be eliminated by treatment in sequence with cyanogen bromide,carboxypeptidase A and carboxypeptidase B to give monomeric Leu¹³!motilin. On both ends of gene 2, there are disposed sites recognizableby the restriction enzymes ClaI and SacI for introduction into vectors.The codons used in gene 2 are mostly those codons that are found withhigh frequency in the genes coding for proteins producible in largequantities in Escherichia coli (M. Goug et al., Nucleic Acids Res., 10,7055 (1982)).

Gene 2 is synthesized by joining the double-stranded DNA fragment fromDNA 3 and DNA 4 to the double-stranded DNA fragment from DNA 5 and DNA 6using ligase. For efficient progress of this ligation reaction, gene 2has been designed such that identical sequences longer than a certainlength be not contained in the base sequence of gene 2.

Step 2 (cf. FIG. 1)

Construction of a plasmid containing gene 2:

The plasmid pTrS20 (prepared by the procedure of Reference Example 1) iscleaved with the restriction enzymes ClaI and SacI and a larger DNAfragment (hereinafter, DNA 7) is isolated. DNA 7 is joined to the geneobtained in step 1 using ligase, whereby the plasmid pMTA1 isconstructed.

Step 3 (cf. FIG. 2)

Construction of a plasmid containing two Leu¹³ !motilin genes:

The plasmid pMTA1 is cleaved with the restriction enzymes PstI and BglIIand a DNA fragment containing the Leu¹³ !motilin gene (hereinafter, DNA8) is isolated. Separately, pMTA1 is cleaved with PstI and BamHI and aDNA fragment containing the Leu¹³ !motilin gene (hereinafter, DNA 9) isisolated. Ligation of DNA 8 with DNA 9 using ligase gives the plasmidpMTA2 containing two Leu¹³ !-motilin genes. The cleavage ends resultingfrom treatment with the restriction enzymes BglII and BamHI areidentical and therefore can be joined together. The ligation site(indicated by Bg/Bam in FIG. 2) is not cleaved with either of therestriction enzymes, however, since it now differs in base sequence fromthe recognition sites for both restriction enzymes.

Step 4 (cf. FIG. 3)

Construction of a plasmid containing four Leu¹³ !motilin genes:

pMTA 2 is cleaved with PstI and BglII. Since, as mentioned above, theBg/Bam site is not cleaved, there is obtained a DNA fragment containingtwo Leu¹³ !motilin genes. Separately, pMTA2 is cleaved with PstI andBamHI to give a DNA fragment containing two Leu¹³ !motilin genes in likemanner. Joining both DNA fragments with ligase gives the plasmid pMTA4containing four Leu¹³ !motilin genes.

The plasmids pMTA8, pMTA16 and pMTA32 which contain eight, sixteen andthirty-two Leu¹³ !motilin genes, respectively, can be prepared in thesame manner as in the construction of pMTA4 from pMTA2. Hereinafter,these are referred to generically as "pMTAs".

Step 5 (cf. FIG. 4)

Insertion of the Leu¹³ !motilin gene into a vector for proteinexpression:

The plasmid psGHIM1 (produced by the procedure of Reference example 2)useful in the expressing of the salmon growth hormone (SGH) gene iscleaved with the restriction enzymes BglII and SacI and a larger DNAfragment (hereinafter, DNA 10) is isolated. Separately, the plasmidpMTA4 is cleaved with BglII and SacI and a DNA fragment containing fourLeu¹³ !motilin genes (hereinafter, DNA 11) is isolated. Joining DNA 10to DNA 11 using ligase gives the expression plasmid pMTK4.

Using pMTA8 or pMTA 16 in lieu of pMTA4 and proceeding in the samemanner as above, one may obtain the expression plasmid pMTK8 or pMTK16containing eight or sixteen Leu¹³ !motilin genes, respectively.Hereinafter, these are referred to generically as "pMTKs".

Step 6 (cf. FIG. 5)

Introduction of a terminator into the expression plasmid:

The plasmid psGHIM1 is cleaved with the restriction enzymes PstI andBamHI and a promoter-containing DNA fragment is recovered. Separately,the plasmid pGHD7 (Reference Example 4) is cleaved with PstI and BamHIto give a terminator-containing DNA fragment. By joining both DNAfragments together using ligase, there is constructed the plasmidpsGHIME1 having no recognition site for the restriction enzyme BglII buthaving an MluI recognition site downstream from the terminator.

The larger DNA fragment obtained by cleavage of psGHIME1 with BglII andSacI is ligated with the Leu¹³ !motilin gene-containing DNA fragmentobtained by cleavage of pMTA4 with BglII and SacI in the presence ofligase to give the plasmid pMTL4, which contains four Leu¹³ !motilingenes. Using pMTK8 or pMTK16 in lieu of pMTA4 and proceeding in the samemanner as above, there is obtained the plasmid pMTL8 or pMTL16containing eight or sixteen Leu¹³ !motilin genes, respectively.Hereinafter, these are referred to generically as "pMTLs".

The plasmids pMTKs and pMTLs each contain a gene located downstream fromthe tryptophan promoter and coding for the 104 amino acids (from theamino terminus) of the salmon growth hormone. Downstream from this gene,a Leu¹³ !motilin gene-containing gene is connected. Further, theterminator for the liporotein gene is introduced downstream from thelast Leu¹³ !motilin gene. The difference between the pMTK plasmids andpMTL plasmids lies in that pMTKs have a recognition site for therestriction enzyme BglII as situated downstream from the terminatorwhile pMTLs do not have such a site.

Step 7

Expression of the Leu¹³ !motilin gene:

The Leu¹³ !motilin gene expression plasmids pMTKs and pMTLs are eachintroduced into Escherichia coli. The transformant produces a fusedprotein composed of Leu¹³ !motilin tetramer, octamer or hexadecamer andthe salmon growth hormone. The thus-produced fused proteins occur asgranules in Escherichia coli cells. The production of the fused proteinderived from Leu¹³ !motilin tetramer is highest and accounts for about10% of the total amount of the cell proteins. The content of Leu¹³!motilin in the granules is 42% for pMTL4, 56% for pMTL8 and 68% forpMTL16.

Step 8 (cf. FIG. 6)

Improvement in the yield of Leu¹³ motilin-(1):

The above method using the pMTK and pMTL plasmids gives Leu¹³ !motilinin the form of the desired peptide fused to the protein portion of thesalmon growth hormone which is composed of 104 amino acids. It isdesirable that this salmon growth hormone portion be as small aspossible. A plasmid in which the salmon growth hormone portion isshorter can be constructed in the manner mentioned below for theenhanced production of Leu¹³ !motilin.

A deoxyoligonucleotide of the formula given below (Formula 13)(hereinafter, DNA 13) and a deoxyoligonucleotide of the formula givenbelow (Formula 14) (hereinafter, DNA 14) are chemically synthesized. Thesynthesis is performed in the manner of solid synthesis by thephosphoramidide method using an automatic DNA synthesizer.

    5' AGCTTATGATAGAAAACCAACGGCTCTTCCA 3' (Formula 13)         SEQ ID NO:8

    5' GATCTGGAAGAGCCGTTGGTTTTCTATCATA 3' (Formula 14)         SEQ ID NO:9

Both the DNAs are mixed together and a linker of the formula given below(Formula 12).

    5' AGCTTATGATAGAAAACCAACGGCTCTTCCA                         SEQ ID NO:8

    3' ATACTATCTTTTGGTTGCCGAGAAGGTCTAG (Formula 12)

Linker 12 contains a gene coding for the amino terminal amino acid tothe 8th amino acid of the salmon growth hormone and, on both sides ofthat gene, the same cohesive ends as produced by the restriction enzymesHindIII and BglII.

The plasmid pMTL4, which contains four Leu¹³ !motilin genes, is cleavedwith the restriction enzymes PstI and BglII, and a Leu¹³ !motilingene-containing DNA fragment (hereinafter, DNA 15) is isolated.Separately, pMTL4 is cleaved with the restriction enzymes PstI andHindIII, and a promoter-containing DNA fragment (hereinafter, DNA 16) isisolated. DNA 15, DNA 16 and linker 12 are joined together using ligase.Thus is constructed the plasmid pMTN4.

The use of pMTL8 in lieu of pMTL4 together with the above procedureleads to the construction of pMTN8. The plasmids pMTN4 and pMTN8 eachcontain a gene coding for a protein composed of the corresponding Leu¹³!motilin polymer and 12 amino acids,Met-Ile-Gln-Asn-Gln-Arg-Leu-Phe-Gln-Ile-Phe-Met, bound to the aminoterminus of that polymer; pMTN4 has four Leu¹³ !motilin genes and pMTN8has eight Leu¹³ !motilin genes. The plasmids pMTN4 and pMTN8 are eachintroduced into Escherichia coli. The resultant transformants producethe desired proteins in almost the same amounts as in the cases of thetransformants carrying the pMTK or pMTL plasmids. Each of the proteinsoccur as granules in Escherichia coli cells. The Leu¹³ !motilin contentin the granules is 77-80% and is much higher than that in the case ofthe pMTK- or pMTL-carrying transformants.

Step 8 (cf. FIG. 7)

Improvement in the yield of Leu¹³ !motilin-(2):

While Leu¹³ !motilin can be produced in large quantities by introducingthe pMTK, pMTL, and pMTN plasmids into Escherichia coli, Leu¹³ !motilincan be produced in higher yields by eliminating a part of the plasmidDNA, as shown below.

The pMTL plasmids have a terminator downstream from the Leu¹³ !motilingenes. A plasmid lacking in the nontranslational (untranslated) regionDNA between this terminator and the last Leu¹³ !motilin gene isconstructed.

Thus, the plasmid pArg4 (Reference Example 5) is cleaved with therestriction enzymes PstI and BamHI and a promoter-containing DNAfragment is recovered. Separately, pGHD7 is cleaved with PstI and BamHIand a terminator-containing DNA fragment is isolated. Both the DNAfragments are joined together using ligase to thereby construct theplasmid pArgE1 which has no BglII recognition site downstream from theterminator.

pArgE1 is cleaved with PstI and EcoRV and a terminator-containing DNAfragment (hereinafter, DNA 17) is isolated.

The plasmid pMTK4 is cleaved with the restriction enzyme SacI, and thesingle-stranded regions corresponding to the cleavage site are renderedblunt-ended by means of hydrolysis with DNA polymerase I, Klenowfragment. The DNA fragment is further cleaved with PstI and a Leu¹³!motilin gene-containing DNA fragment (hereinafter, DNA 18) is isolated.Joining DNA 17 to DNA 18 using ligase gives the plasmid pMTM4.

Using pMTL4 instead of the plasmid pMTK4 and proceeding in the samemanner as above, pMTM4 is obtained.

Using mMTL8 instead of pMTK4 and proceeding in the same manner as above,the plasmid pMTM8, which contains eight Leu¹³ !motilin genes isobtained.

pMTM8 can be constructed from pMTM4 in the same manner as in theconstruction of pMTA8 from-pMTA4. Thus, pMTM4 is cleaved with therestriction enzymes PstI and BglII and a Leu¹³ !motilin gene-containingDNA fragment is isolated. This DNA fragment is joined, in the presenceof ligase, to the Leu¹³ !motilin gene-containing DNA fragment obtainedby cleavage of pMTM4 with PstI and BamHI, to give pMTM8.

Furthermore, using pARG4 in lieu of the plasmid pArgE1 and proceeding inthe same manner as in the construction of pMTM4 or pMTM8, the plasmidspMTm4 and pMTm8 are constructed. The difference between pMTMs and pMTmslies in that pMTms have a recognition site for the restriction enzymeBglII while pMTMs have no such site.

Step9

Expression of the Leu¹³ !motilin gene using pMTMs:

The pMTM and pMTm plasmids have a structure such that about 160 basepairs (hereinafter, bp) of the nontranslational region gene upstreamfrom the terminator have been eliminated from pMTKs and pMTLs.

When pMTM4 is introduced into Escherichia coli HB101, the Leu¹³ !motilinpolymer protein content accounts for 17% of the total protein content.

Step 10 (cf. FIG. 8)

Improvement in the yield of Leu¹³ !motilin-(3):

The pMTN plasmids have a terminator downstream from the Leu¹³ !motilingene region. Plasmids, in which the nontranslational region gene betweenthe terminator and the Leu¹³ !motilin gene region has been eliminated,are constructed as follows:

The plasmid pArgE1 is cleaved with PstI and EcoRV and aterminator-containing DNA fragment (hereinafter, DNA 19) is isolated.The plasmid pMTN4 is cleaved with the restriction enzyme SacI, and thesignle-stranded regions corresponding to the cleavage site are convertedto blund ends by hydrolysis using DNA polymerase I, Klenow fragment.After further cleavage with the restriction enzyme PstI, a Leu¹³!motilin gene-containing DNA fragment (hereinafter, DNA 20) is isolated.Ligation of both DNA fragments using ligase gives the plasmid pMTO4.

pMTO8 is constructed using pMTN8 in lieu of pMTN4 and following theabove procedure. Like pMTM8, pMTO8 can be prepared also by the methodused in constructing pMTA8 from pMTA4.

A series of pMTo plasmids are constructed in the same manner as aboveusing pArg4 in lieu of pArgE1. The pMTO plasmids differ from the pMToplasmids in that the latter have a recognition site for the restrictionenzyme BglII downstream from the terminator while the former have nosuch site.

The pMTO and pMTo plasmids have a structure such that about 160 bp ofthe nontranslational region gene upstream from the terminator have beeneliminated from pMTNs. pMTO4 is introduced into Escherichia coli tocause protein expression. The transformant produces the protein equallyin large amounts as is the case with pMTN4. The protein occurs asgranules in the transformant strain, and the Leu¹³ !motilin content inthe granules is 77%.

Step 11

Promoter modification:

The plasmid pMTO4 contains two tryptophan promoters coupled in series(Ptrp×2) and the distance between the Shine-Dalgarno (SD) sequence andthe initiation codon (ATG) (SD-ATG) corresponds to 10 bases. The plasmidpMTO4 is cleaved with the restriction enzymes HindIII and PstI and aLeu¹³ !motilin gene-containing DNA fragment (hereinafter, DNA 2i) isisolated. Separately, the plasmid pKYP10 (European Patent PublicationNo. 83069A) is cleaved with the restriction enzymes HindIII and PstI anda promoter-containing DNA fragment (hereinafter, DNA 22) is isolated.Ligation of DNA 21 with DNA 22 using ligase gives the plasmid pMTOI4(cf. FIG. 9). The plasmid pMTOI4 contains one tryptophan promoter (Ptrp)and the SD-ATG distance therein corresponds to 14 bases.

The plasmid pGEL1 (European Patent Publication No. 166444A; FERMBP-612), which is used in lieu of the plasmid pKYP10, is cleaved withthe restriction enzymes HindIII and PstI and a promoter-containing DNAfragment (hereinafter, DNA 23) is isolated. DNA 21 (obtained from theplasmid pMTO4) and DNA 23 are joined together using ligase, whereby theplasmid pMTOII4 is obtained (cf. FIG. 10). The plasmid pMTOII4 containstwo promoters (Ptrp×2) and the SD-ATG distance therein amounts to 14bases.

The plasmid pGHA2 (European Patent Publication No. 152613A; IGHA2; FERMBP-400), which is used in lieu of the plasmid pGEL, is cleaved with therestriction enzymes HindIII and PstI and a promoter-containing DNAfragment (hereinafter, DNA 24) is isolated. DNA 21 (obtained from theplasmid pMTO4) and DNA 24 are joined together using ligase to give theplasmid pMTOIII4 (cf. FIG. 11). The plasmid pMTOIII4 contains a letpromoter (Plet) and the SD-ATG distance therein is 14 bases long.

Each Leu¹³ !motilin polymer produced abundantly by virtue of the abovegenetic engineering techniques is accumulated, in the form of granules,in bacterial cells. The cells are disrupted and then subjected tocentrifugation, whereby the granules are readily separated from themembrane components and soluble fractions. The Leu¹³ !motilin polymer isthus obtained in good yields and in high purity. When this granularLeu¹³ !motilin polymer is treated with cyanogen bromide for degradation,cleavage takes place at the site of the methionine in the spacerpeptide, giving a peptide (peptide 25) composed of 26 amino acids,namely monomeric Leu¹³ !motilin and Arg-Ile-Phe-Hse (methionine havingbeen converted to homoserine (Hse) as a result of degradation) bound tothe carboxyl side of said Leu¹³ !motilin. Digestion of this peptide 25with carboxypeptidase A results in hydrolytic elimination from thecarboxyl side of Hse, Phe and Ile in that order. Thus is obtained apeptide (peptide 26) composed of Leu¹³ !motilin and Arg bound to thecarboxyl terminus thereof as a single product.

The subsequent digestion of peptide 26 with carboxypeptidase Beliminates Arg to give Leu¹³ !motilin (Formula 1) as a single product inhigh yields. ##STR3##

As mentioned above, the production method according to the inventiongives Leu¹³ !motilin in very high yields by virtue of the geneticengineering techniques. The production method according to the inventionconsists in construction of a gene coding for a peptide polymer composedof a plurality of molecules of the desired peptide connected in serieswith a spacer peptide, which is eliminable chemically and enzymatically,inserted between every two molecules of the desired peptide; insertionof the gene into a plasmid having an effective promoter and aterminator; introduction of the resultant plasmid into a microorganismto thereby cause production of the peptide polymer in significantquantities; conversion of the thus-obtained peptide polymer to thedesired monomeric peptide in high yields by chemical and enzymatictreatment; and recovering the desired peptide.

While the use of cyanogen bromide, carboxy peptidase A andcarboxypeptidase B for spacer peptide elimination has been mentioned inthis disclosure, any other elimination method can be employed providedthat method used will not cause decomposition of the desired peptide.The use of formic acid, for instance, for chemical degradation, resultsin cleavage of the Asp-Pro bond, whereas hydroxylamine cleaves the Asn-X(X being Gly, Leu or Ala) bond. For enzymatic degradation, the use ofenterokinase for cleaving the (Asp)_(n) -Lys (n=2 to 4) bond, the use ofcollagenase for cleaving Pro-X-Gly-Pro (X being any amino acid residue)and the use of kallikrein for cleaving the Phe-Arg bond may bementioned, among others.

The spacer peptide to be employed in practicing the invention may haveany structure that can be cleaved by a cleavage method which will notcause cleavage of the desired peptide and which is selected from amongthe cleavage methods as mentioned above, provided that the resultantspacer peptide fragment or fragments can be eliminated by a suitablemethod or methods. In processes for producing peptide monomers whichinvolve production of polymers containing peptide monomers connected inseries and cleavage of said polymers, the use of a spacer peptide isgenerally essential. The use of a spacer peptide is unnecessary onlywhen a selective method is available for cleaving the bond between theamino terminus and carboxyl terminus of the peptide to be produced. Forinstance, in cases where the amino terminus is Pro and the carboxylterminus is Asp, the peptide polymers in which monomer units areconnected via the bond Asp-Pro can be converted to the monomeric form bytreatment with formic acid. However, peptides having such structure arevery scarce. Accordingly, spacer peptides are generally essential andthey must be designed so that they can be cleaved and eliminated readilyand in high yields.

The spacer peptide specifically disclosed herein has wide application asit can be used in producing all peptides that are free of methionine.Thus, when the carboxyl-terminal amino acid of the desired peptide isother than a basic amino acid, the spacer peptide according to theinvention can be used and can be cleaved and eliminated as well by themethod specifically disclosed herein. When the carboxyl-terminal aminoacid of the desired peptide is the basic amino acid Lys or Arg, thebasic amino acid Arg in the spacer peptide according to the invention isunnecessary and the reaction for spacer peptide elimination followingcleavage with cyanogen bromide needs only carboxypeptidase A;carboxypeptidase B is unnecessary.

The gene coding for the spacer peptide according to the invention hasrecognition sites for the restriction enzymes BglII and BamHI and theutility of the method comprising combining, by means of said gene, anumber of genes treated with enzymes producing the same cleavage terminihas been demonstrated above. In combining a number of genes in series, anumber of enzymes can be used in addition to the combination of BglIIand BamHI which combination is preferably used in accordance with theinvention. The only requirement is that the restriction enzymes usedgive cleavage sites to the base sequence coding for a spacer peptidewhich meets the above-mentioned conditions relative to cleavage andelimination.

While the gene production method has been described herein relative togenes containing 2^(n) (n=1 to 5) Leu¹³ !motilin genes, namely 1, 2, 4,8, 16 and 32 Leu¹³ !motilin genes, joined together in series,respectively, it is evident that any optional number of genes coding forthe peptide according to the invention can be connected with one anotherin series with ease by the relevant method disclosed herein. Althoughmention has been made herein of the production of Leu¹³ !motilinpolymers in the form of fused proteins resulting from the connection ofthe genes coding for the polymers to a part of the gene for the aminoterminal side of the salmon growth hormone or IFN-γ at a site downstreamthereof, it will be understood that that part of salmon growth hormoneor IFN-γ gene is not an essential element in producing the desired Leu¹³!motilin polymers.

However, the base sequence in the vicinity of the initiation codon ATGof a gene has a great influence on the protein yield. The use of genesthat have the base sequence around the initiation codon ATG of thesalmon growth hormone gene, which is known to give high productivity,can result in the high-level expression of the Leu¹³ !motilin polymergenes. This method also allows high-level expression of genes forpeptide polymers other than Leu¹³ !motilin polymers as well andaccordingly has considerable general applicability beyond the specificsof making the Leu¹³ !motilin, as will be apparent.

The reaction conditions to be employed in the above recombinant DNAtechniques are consistent with the usual techniques and are generally asfollows:

The reaction for DNA digestion with a restriction enzyme or enzymes iscarried out generally in a reaction mixture composed of 0.1-20 μg of DNAand a medium containing 2-200 mM (preferably 10-40 mM) Tris-HCl (pH6.0-9.5, preferably pH 7.0-8.0), 0-200 mM NaCl or KCl, 2-30 mM(preferably 5-10 mM) MgCl₂ and 0-20 mM mercaptoethanol, with 0.1-100units (preferably 1-3 units per microgram of DNA) of a restrictionenzyme or enzymes added, at 20°-70° C. (the optimal temperature may varydepending on the restriction enzyme or enzymes used) for 15 minutes to24 hours.

The DNA fragment formed upon restriction enzyme digestion is purified bythe LGT method or by polyacrylamide gel electrophoresis, for instance.

The reaction for ligating DNA fragments is carried out in a reactionmedium containing 2-200 mM (preferably 10-40 mM) Tris-HCl (pH 6.1-9.5,preferably pH 7.0-8.0), 2-20 mM (preferably 5-10 mM) MgCl₂, 0.1-10 mM(preferably 0.5-2.0 mM) ATP and 1-50 mM (preferably 5-10 mM)dithiothreitol, using 0.3-10 units of T4 DNA ligase, at 1°-37° C.(preferably 3°-20° C.) for 15 minutes to 72 hours (preferably 2-20hours).

The recombinant plasmid DNA formed by the ligation reaction isintroduced into Escherichia coli, as necessary by using thetransformation method of Cohen et al. (S. N. Cohen et al., Proc. Natl.Acad. Sci. USA, 69, 2110 (1972)).

The DNA isolation from the Escherichia coli strain harboring therecombinant plasmid DNA is carried out by the cesium chloride-ethidiumbromide density gradient ultracentrifugation method (D. B. Clewell etal., Proc. Natl. Acad. Sci. USA, 62, 1159 (1969)) or by the method ofBirnboim et al. (H. C. Birnboim et al., Nucleic Acids Res., 7, 1513(1979)), for instance.

The plasmid DNA is digested with an appropriate restriction enzyme orenzymes and examined for the cleavage site or sites by agarose gelelectrophoresis or polyacrylamide gel electrophoresis. If necessary, thebase sequence of the DNA is further determined by the Maxam-Gilbertmethod (Proc. Natl. Acad. Sci., USA, 74, 560 (1977)) or by the Sanger'smethod which uses M13 phage (Sanger et al., Proc. Natl. Acad. Sci., USA,74, 5463 (1977); Amersham's M13 Cloning and Sequencing Handbook).

The peptide according to the invention can be produced in the followingmanner:

Escherichia coli K-12 C600 or HB101, for instance, is transformed withthe plasmid and plasmid-carrying Escherichia coli transformants areselected from among ampicillin-resistant colonies. By cultivating theplasmid-carrying Escherichia coli strains in a medium, peptide can beproduced in the culture.

Any medium, either synthetic or natural, may be used as the growthmedium, provided that it is suited for the growth of Escherichia coliand for the production of the peptide.

Usable as the carbon sources are glucose, fructose, lactose, glycerol,mannitol and sorbitol, among others. Usable as the nitrogen sources are,for example, NH₄ Cl, (NH₄)₂ SO₄, casamino acids, yeast extract,polypeptone, meat extract, Bactotryptone and corn steep liquor. K₂ HPO₄,KH₂ PO₄, NaCl, MgSO₄, vitamin B₁, MgCl₂, and so forth can be used asother nutrient sources.

The cultivation is carried out with aeration and stirring at a pH of 5.5to 8.5 and at a temperature of 18°-40° C.

After 5-90 hours of cultivation, the peptide accumulated in culturedcells is recovered by harvesting cells from the culture, treating thesame with lysozyme, disrupting the same by repeated freezing-thawingcycles, and extracting the peptide from the supernatant obtained bycentrifugation by a conventional method of peptide extraction.

The peptide can be detected by directly dissolving the cultured cells inLaemmli's sample buffer (Laemmli, Nature, 227, 680 (1970)) with heating,and applying to SDS-polyacrylamide gel electrophoresis (Laemmli'smethod; vide supra), followed by straining with the dye CoomassieBrilliant Blue (Bio-Rad).

The following examples are further illustrative of the presentinvention.

EXAMPLE 1

Production of DNAs 3-6, 13 and 14:

DNAs 3-6, 13 and 14 were synthesized by phosphoramidide method in themanner of solid phase synthesis L. Beaucage et al., Tetrahedron Letters,22, 1859 (1981); L. J. McBrie et al., ibid., 24, 245 (1983)) using anApplied Biosystems model 380A automatic DNA synthesizer, as follows:

Silica gel was used as the solid-phase carrier. (1) A nucleotide wascondensed with the 5' hydroxyl group of a nucleotide bound to thesolid-phase carrier via the 3'-hydroxyl group thereof by thephosphoramidide method, (2) the phosphite bond in the condensednucleotide was oxidized in a phosphate bond with iodine, and (3) theprotective group on the 5' hydroxyl group of the condensed nucleotidewas removed with trifluoroacetic acid. Then, step (1) was repeated forthe condensation of the next nucleotide. In this way, steps (1)-(3) wererepeated, and a DNA was synthesized on the carrier. After completion ofthe synthesis, the carrier with the DNA bound thereto was allowed tostand in a thiophenol solution for 1 hour at room temperature to therebycause elimination of the protective group on the phosphoric acid moietyand then allowed to stand in concentrated aqueous ammonia for 1 hour atroom temperature, whereby the DNA was released from the carrier. TheDNA-containing concentrated aqueous ammonia was heated at 60° C. in asealed vessel for 12 hours to eliminate the protective groups on thebases.

In the case of DNA 3, for instance, the synthesis was carried out using1 μM of the starting nucleotide bound to the carrier. After completionof the last condensation reaction in an overall yield of 81% and thesubsequent deprotection and release from the solid phase, there wasobtained DNA 3 as a crude product in a yield of 242 O.D. units (measuredat 260 nm). For purification, 22 O.D. units of this crude product waselectrophoresed on a 10% polyacrylamide gel (2 mm thick, 13 cm×13 cm)using tris-borate buffer (pH 8) containing 7M urea. That portion of thegel which contained DNA 3 was collected and extracted with 1 ml of 0.2Mtriethylamine carbonate buffer (pH 8) (hereinafter, TEAB) for 18 hours.The extract was applied to a SEPHADEX DE52® (Pharmacia Fine Chemicals)absorption column (6 mm in diameter, 5 mm in length) for causing DNA 3to be adsorbed thereon. Elution with 2 ml of 2M TEAB gave 3.9 O.D. unitsof pure DNA 3.

Other DNAs than DNA 3 were also synthesized in almost the same yields.

These DNAs were radiolabeled by phosphorylating them on the 5'-hydroxylgroup thereof by the conventional method (A. M. Maxam et a., Methods inEnzymology, vol. 65, part I, p. 499, Academic Press (1980)) using phageT4 nucleotide kinase and γ-³² P!ATP. The labeled DNAs were subjected to20% polyacrylamide gel electrophoresis using tris-borate buffercontaining 7M urea. In this way, the purity and chain length of each DNAwas confirmed. Furthermore, the base sequence of each labeled DNA wasdetermined by the Maxam-Gilbert method (vide supra) and it was confirmedthat each DNA had the respective desired base sequence.

EXAMPLE 2

Construction of the plasmid pMTA1:

The plasmid pTrS20 (2 μg) was dissolved in 30 μl of a solution (10 mMTris-HCl (pH 7.5), 7 mM MgCl₂, 6 mM 2-mercaptoethanol) containing 10units of the restriction enzyme ClaI (Boehringer Mannheim) and 15 unitsof the restriction enzyme SacI (Takara Shuzo), and the digestionreaction was carried out at 37° C. for 2 hours. The reaction mixture waselectrophoresed on an ethidium bromide-containing agarose gel. Underdetection with ultraviolet rays at a wavelength of 302 nm, a gel piececontaining DNA 7 of about 3.8 kb was excised. To the gel piece was added0.5 ml of phenol, the mixture was frozen and thawed, the aqueous layerwas washed with chloroform, and the DNA was recovered by precipitationwith ethanol.

DNAs 3-6 (each 10 picomoles) each was dissolved in 30 μl of a buffer forT4 polynucleotide kinase reaction (50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂,5 mM dithiothreitol (hereinafter, DTT), 1 mM ATP, 0.1 mM spermidine, 0.1mM EDTA). Following addition of 3 units of T4 polynucleotide kinase(Takara Shuzo), the phosphorylation reaction was carried out at 37° C.for 40 minutes. Thereafter, the enzyme was inactivated by heating at 65°C. for 15 minutes. A 4-μl portion was taken from each reaction mixturethus obtained. The 4-μl portions from the four reaction mixtures werecombined, 0.08 picomole of DNA 7 (obtained in the above manner) wasadded. The volume of the mixture was increased to 50 μl, with additionof 2 units of T4 DNA ligase (Takara Shuzo), while the mixturecomposition was adjusted to: 28 mM Tris-HCl (pH 7.5), 9 mM MgCl₂, 10 mMDTT, 0.03 mM EDTA, 0.7 mM ATP and 0.03 mM spermidine. The ligationreaction was effected at 4° C. for 16 hours.

The reaction mixture was used to transform the Escherichia coli HB101strain (Bolivar et al., Gene, 2, 75 (1977)) by the method of Cohen et al(S. N. Cohen et al., Proc. Natl. Acad. Sci. USA, 69 2110 (1972)), andampicillin-resistant (Ap^(r)) colonies were obtained. The plasmid DNAwas recovered from one of the colonies by the alkali treatment method(Maniatis et a. (ed.), Molecular Cloning, p. 368, Cold String HarborLaboratory). Thus was obtained pMTA1. The structure of pMTA1 wasconfirmed by cleavage with BglII, PstI, SacI and BamHI, followed byagarose gel electrophoresis. For each cleavage reaction in the presenceof the enzyme concerned, a reaction medium was prepared by adding NaClor KCl in an optimal concentration for said enzyme as selected withinthe range of 0-200 mM to a solution containing 10 mM Tris-HCl (pH 7.5).,7 mM MgCl₂ and 6 mM 2-mercaptoethanol hereinafter, restriction enzymereaction medium; the NaCl or KCl concentration alone being givenhereinafter).

Furthermore, the desired Leu¹³ !motilin gene was identified bydetermining the 115-base sequence covering the portions derived fromDNAs 3-6 by the method described in the literature (A. J. H. Smith,Methods in Enzymology, edited by L. Grossmen and K. Moldave, vol. 65, p.560 (1980), Academic Press).

EXAMPLE 3

Construction of the plasmid pMTA2:

A 0.2-μg portion of the plasmid pMTA1 obtained in Example 2 wasdissolved in 15 μl of the restriction enzyme reaction medium (100 mMNaCl) defined in Example 2, 6 units of PstI (Takara Shuzo) and 6 unitsof BglII (Toyo Jozo) were added, and the digestion reaction was effectedat 37° C. for 2 hours. The reaction mixture was fractionated by the sameagarose gel electrophoresis as carried out in Example 2. Thus wasobtained a DNA fragment (DNA 8) of about 2.8 kb.

Separately, 0.2 μg of pMTA1 was dissolved in 15 μl of the restrictionenzyme reaction medium (100 mM KCl), 6 units of PstI and 6 units ofBamHI (Takara Shuzo) were added, and the digestion reaction was effectedat 37° C. for 2 hours. Fractionation of the reaction mixture by agarosegel electrophoresis gave a DNA fragment (DNA 9) about 1.2 kb in length.

The thus-obtained DNA 8 (50 ng) and DNA 9 (50 ng) were combined anddissolved in 30 μl of a medium for T4 DNA ligase reaction (20 mMTris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 0.3 mM ATP), 1 unit of T4 DNAligase (Takara Shuzo) was added, the the ligation reaction was effectedat 4° C. for 18 hours. The reaction mixture was used to transform theEscherichia coli HB101 strain, and Ap^(r) colonies were isolated. Theplasmid DNA was recovered from one of said colonies. Thus was obtainedpMTA2. The structure of this plasmid was confirmed by cleavage withPstI, BamHI and BglII, followed by agarose gel electrophoresis.

EXAMPLE 4

Construction of the plasmid pMTA4:

A 0.2-μg portion of the plasmid pMTA2 obtained in Example 3 wasdissolved in 15 μl of the restriction enzyme reaction medium (100 mMNaCl) defined in Example 2, 6 units of PstI and 6 units of BglII wereadded, and the plasmid was digested at 37° C. for 2 hours. Fractionationby agarose gel electrophoresis gave a DNA fragment about 2.8 kb inlength. Separately, 0.2 μg of pMTA2 was dissolved in 15 μl of therestriction enzyme reaction medium (100 mM KCl), 6 units of PstI and 6units of BamHI were added and digestion was effected at 37° C. for 2hours. Fractionation by agarose gel electrophoresis gave a DNA fragmentabout 1.3 kb in length.

The above two DNA fragment (each 8.2 picomole) were combined anddissolved in 30 μl of the T4 DNA ligase reaction medium, i unit of T4DNA ligase was added, and the ligation reaction was effected at 4° C.for 18 hours. The reaction mixture was used to transform the Escherichiacoli HB101 strain, Ap^(r) colonies were isolated, the the plasmid DNAwas recovered. The structure of the thus-obtained plasmid pMTA4 wasconfirmed by the cleavage with PstI, BamHI and BglII, followed byagarose gel electrophoresis.

EXAMPLE 5

Construction of the plasmid pMTL4:

A 1-μg portion of the plasmid psGHIM1 was dissolved in 30 μl of therestriction enzyme reaction medium (100 mM KCl), 6 units of PstI and 6units of BamHI were added, and the digestion reaction was effected at37° C. for 2 hours. A DNA fragment of about 2.1 kb was recovered byagarose gel electrophoresis.

The plasmid pGHD7 (1 μg) was digested with PstI and BamHI in the samemanner as in the case of psGHIM1, and a DNA fragment of about 1.7 kb wasrecovered.

These DNA fragment (each 0.03 picomole) were combined and dissolved in30 μl of the T4 DNA ligase reaction medium, 1 unit of T4 DNA ligase wasadded, and the ligation reaction was effected at 4° C. for 18 hours. Thereaction mixture-was used to transform the Escherichia coli HB101strain, Ap^(r) colonies were isolated, and the plasmid DNA wasrecovered. Thus was obtained psGHIME1.

A 0.5-μg portion of the thus-obtained plasmid psGHIME1 was dissolved in30 μl of the restriction enzyme reaction medium (neither NaCl nor KCladded), 50 units of SacI (Takara Shuzo) was added, and the digestionreaction was effected at 37° C. for 2 hours. Then, 2 μl of 2M NaCl wasadded and further 15 units of BglII was added, and the digestionreaction was continued at 37° C. for additional 2 hours. A DNA fragmentof about 3.2 kb was recovered following fractionation by agarose gelelectrophoresis.

A 0.5-μg portion of pMTA4 (obtained in Example 4) was digested in thesame manner as above with SacI and then with BglII, and a DNA fragmentof about 0.3 kb was recovered by agarose gel electrophoresis.

These DNA fragments (each 0.03 picomole) were combined and dissolved in30 μl of the T4 DNA ligase reaction medium, 1 unit of T4 DNA ligase wasadded, and the ligation reaction was effected at 4° C. for 15 hours. Thereaction mixture was used to transform the Escherichia coli HB101strain, Ap^(r) colonies were isolated, and the plasmid DNA wasrecovered. Thus was obtained pMTL4.

EXAMPLE 6

Construction of the plasmid pMTN4:

A 1.5-μg portion of the plasmid pMTL4 obtained in Example 5 wasdissolved in 20 μl of the restriction enzyme reaction medium (100 mMNaCl), 8 units of PstI and 7 units of BglII were added, and thedigestion reaction was effected at 37° C. for 2 hours. A DNA fragment ofabout 2.2 kb (DNA 15) was recovered by agarose gel electrophoresis.Separately, pMTL4 was dissolved in 20 μl of the restriction enzymereaction medium (100 mM NaCl), 8 units of PstI and 6 units of HindIII(Takara Shuzo) were added, and the digestion reaction was effected at37° C. for 2 hours. Following agarose gel electrophoresis, a DNAfragment of about 1.0 kb (DNA 16) was recovered.

DNA 15 (21 picomoles) and DNA 16 (21 picomoles) were each dissolved in50 μl of the T4 polynucleotide kinase reaction buffer defined in Example2, 4 units of T4 polynucleotide kinase was added, and thephosphorylation reaction was effected at 37° C. for 40 minutes. Theenzyme was then inactivated by heating at 65° C. for 15 minutes. Thereaction mixtures of (2 μl each) were combined, DNA 15 and DNA 16 wereadded, and the mixture was made 40 μl while adjusting the composition asfollows, with addition of 2 units of T4 DNA ligase: 28 mM Tris-HCl, 9 mMMgCl₂, 10 mM DTT, 0.03 mM EDTA, 0.7 mM ATP, 0.03 mM spermidine. Theligation reaction was then effected at 4° C. for 16 hours. The reactionmixture was used to transform the Escherichia coli HB101 strain, Ap^(r)colonies were isolated, and the plasmid DNA was recovered. Thus wasobtained pMTN4.

EXAMPLE 7

Construction of the plasmid pMTO4:

A 2.5-μg portion of the plasmid pMTN4 was dissolved in 40 μl of therestriction enzyme reaction medium (neither NaCl nor KCl added)containing 0.01% of TRITON nonionic surfactant 12 units of SacI wasadded, and the digestion reaction effected at 37° C. for 2 hours. Thereaction mixture was extracted with phenol-chloroform, and SacI-cleavedpMTN4 was recovered by precipitation with ethanol and dissolved in 40 μlof a solution having the composition: 20 mM Tris-HCl (pH 7.8), 7 mMMgCl₂, 6 mM 2-mercaptoethanol, 0.25 mM each dNTP (dATP, dTTP, dCTP,dGTP). To the solution was added 4 units of Escherichia coli DNApolymerase I, Klenow fragment (Takara Shuzo), and the reaction waseffected at 20° C. for 1 hour. The reaction mixture was extracted withphenol-chloroform, and polymerase-treated pMTN4 fragment was recoveredby precipitation with ethanol. This was dissolved in 30 μl of therestriction enzyme reaction medium (100 mM NaCl), 5 units of PstI wasadded, and the digestion reaction was effected for 2 hours.Fractionation by agarose gel electrophoresis gave a DNA fragment ofabout 1.3 kb (DNA 20).

A 2-μg portion of the plasmid pArgE1 was dissolved in 20 μl of therestriction enzyme reaction medium (150 mM NaCl), 10 units of PstI and10 units of EcoRV (Takara Shuzo) were added, and the digestion reactionwas effected at 37° C. for 2 hours. Fractionation by agarose gelelectrophoresis gave a DNA fragment Of about 1.7 kb (DNA 19).

This DNA 19 (0.06 picomole) and DNA 20 (0.06 picomole) were combined anddissolved in 25 μl of the T4 DNA ligase reaction medium, 3 units of T4DNA ligase was added, and the ligation reaction was effected at 4° C.for 20 hours. The reaction mixture was used to transform the Escherichiacoli HB101 strain, Ap^(r) colonies were isolated, and the plasmid DNAwas recovered from one of the colonies. Thus was obtained pMTO4. Thestructure of pMTO4 was confirmed by cleavage with PstI, EcoRI, HindIII,SalI, BglII and MluI.

EXAMPLE 8

Construction of the plasmid pMTOI4:

The plasmid pMTO4 (2 μg) was dissolved in 30 μl of the restrictionenzyme reaction medium (100 mM NaCl), 6 units of HindIII and 6 units ofPstI were added, and the digestion reaction was effected at 37° C. for 2hours. A Leu¹³ !motilin polymer gene-containing DNA fragment of about3.0 kb (DNA 21) was recovered by fractionation by agarose gelelectrophoresis. Separately, 3 μg of the plasmid pKYP10 (European PatentPublication No. 83069A) was dissolved in 30 μl of the restriction enzymereaction medium (100 mM NaCl), 9 units of HindIII and 9 units of PstIwere added, the digestion reaction was effected at 37° C. for 2 hours,and a promoter-containing DNA fragment of about 1.1 kb (DNA 22) wasrecovered by fractionation by agarose gel electrophoresis. These DNAfragments (about 0.1 mg each) were dissolved in 30 μl of the T4 DNAligase reaction medium, 1 unit of T4 DNA ligase was added, and theligation reaction was effected at 4° C. for 18 hours. The reactionmixture was used to transform the Escherichia coli HB101 strain, and theplasmid DNA was recovered from one of the Ap^(r) colonies obtained. Thuswas obtained the Leu¹³ !motilin polymer expression plasmid pMTOI4containing a tryptophan promoter. The structure of pMTOI4 was confirmedby cleavage with PstI, BanIII, HindIII and MluI (cf. FIG. 9).

EXAMPLE 9

Construction of the plasmid pMTOII4:

The plasmid pMTO4 (2 μg) was dissolved in 30 μl of the restrictionenzyme reaction medium (100 mM NaCl), 6 units of HindIII and 6 units-ofPstI were added, and the digestion reaction was effected at 37° C. for 2hours. A Leu¹³ !motilin polymer gene-containing DNA fragment of about3.0 kb (DNA 21) was recovered by fractionation by agarose gelelectrophoresis. Separately, 3 μg of the plasmid pGEL1 (European PatentPublication No. 166444A) was dissolved in 30 μl of the restrictionenzyme reaction medium (100 mM NaCl), 9 units of HindIII and 9 units ofPstI were added, and the digestion reaction was effected at 37° C. for 2hours. A promoter-containing DNA fragment of about 1.1 kb (DNA 23) wasrecovered by fractionation by agarose gel electrophoresis. These DNAfragments (about 0.1 μg each) were dossolved in 30 μl of the T4 DNAligase reaction medium, 1 unit of T4 DNA ligase was added, and theligation reaction was effected at 4° C. for 18 hours. The reactionmixture was used to transform the Escherichia coli HB101 strain, and theplasmid DNA was recovered from one of the Ap^(r) colonies obtained. Thuswas obtained the plasmid pMTOII4 for Leu¹³ !motilin polymer expression.The plasmid contained two tryptophan promoters coupled in series and, inthe plasmid, the SD sequence was 14 bases apart from the initiationcodon ATG. The structure of pMTOII4 was confirmed by cleavage with PstI,BanIII, HindIII and MluI (cf. FIG. 10).

EXAMPLE 10

Construction of the plasmid pMTOIII4:

The plasmid pMTO4 (2 μg) was dissolved in 30 μl of the restrictionenzyme reaction medium (100 mM NaCl), 6 units of HindIII and 6 units ofPstI were added, and the digestion reaction was effected at 37° C. for 2hours. Following fractionation by agarose gel electrophoresis, a Leu¹³!motilin polymer gene-containing. DNA fragment of about 3.0 kb wasrecovered. Separately, 3 μg of the plasmid pGHA2 (European PatentPublication No. 152613A) was dissolved in 30 μl of the restrictionenzyme reaction medium (100 mM NaCl), 9 units of HindIII and 9 units ofPstI were added, and the digestion reaction was effected at 37° C. for 2hours. Following fractionation by agarose gel electrophoresis, apromoter-containing DNA fragment of about 0.9 kb was recovered. TheseDNA fragments (about 0.1 μg each) were dissolved in 30 μg of the T4 DNAligase reaction medium, 1 unit of T4 DNA ligase was added, and theligation reaction was effected at 4° C. for 18 hours. The reactionmixture was used to transform the Escherichia coli HB101 strain, the theplasmid DNA was recovered from one of the Ap^(r) colonies obtained. Thuswas obtained the plasmid pMTOIII4 for Leu¹³ !motilin polymer expression,which contained a let promoter. The structure of pMTOIII4 was confirmedby cleavage with PstI, BanIII, HindIII, MluI and BglII (cf. FIG. 11).

EXAMPLE 11

Production of a Leu¹³ !motilin polymer protein in Escherichia coli usingthe plasmid pMTO4:

The Escherichia coli W3110 strA strain (FERM BP-732) was transformedwith pMTO4 (obtained in Example 7). An Ap^(r) colony thus obtained wasinoculated into 8 ml of LG medium (1% Bacto-tryptone, 0.5% yeastextract, 0.5% NaCl, 0.1% glucose, 50 μg/ml tryptophan, 50 μg/mlampicillin, pH 7.5) and cultured at 30° C. for 16 hours. A 400-μlportion of the culture was inoculated into 10 ml of MEG medium (0.6% Na₂HPO₄, 0.3% KH2PO₄, 0.5% NaCl, 0.1% NH₄ Cl, 0.5% glucose, 0.5% casaminoacids, 1 mM MgSO₄, 4 μg/ml vitamin B1, pH 7.2) supplemented with 50μg/ml tryptophan and 50 μg/ml ampicillin and cultured at 30° C. When theturbidity (OD₅₅₀) of the culture had reached 0.9 (after about 4 hours),200 μg of indoleacrylic acid was added. The cultivation was continuedfor a further 4 hours. Then, cells were recovered by centrifuging theculture at 7,000 rpm for 5 minutes. The cells were dissolved in thesample buffer of Laemmli et al. (Nature, 227, 680 (1970)), and thesolution was heated and subjected to SDS-polyacrylamide gelelectrophoresis after the method of Laemmli et al. As a result ofstaining with Coomassie Brilliant Blue, a polypeptide band was detectedat a position corresponding to a molecular weight of about 15,000. theEscherichia coli W3110 strA strain free from pMTO4 gave no correspondingband. It was thus established that the pMTO4-carrying transformant ofEscherichia coli W3110 strA had produced a Leu¹³ !motilin polymerprotein as fused with a part of the salmon growth hormone.

EXAMPLE 12

Production of a Leu¹³ motilin polymer protein in Escherichia coli usingthe plasmid pMTOI4:

The Escherichia coli W3110 strA strain transformed with the plasmidpMTOI4 obtained in Example 8. An Ap^(r) colony obtained was cultured inthe same manner as in Example 11, and cells were recovered bycentrifuging the culture at 7,000 rpm for 5 minutes. The cells weredissolved in the sample buffer according to Laemmli et al., and thesolution was heated and subjected to SDS-polyacrylamide gelelecrtrophoresis after the method of Laemmli et al., followed byCoomassie Brilliant Blue staining. As a result, a polypeptide band wasdetected at a position corresponding to a molecular weight of about15,000. Since the pMTOI4-free Escherichia coli W3110 strA strain gave nocorresponding band, it was evident that the pMTOI4-carrying transformantof Escherichia coli W3110 strA had produced a Leu¹³ !motilin polymerprotein as fused with a part of the salmon growth hormone.

EXAMPLE 13

Production of a Leu¹³ !motilin polymer protein in Escherichia coli usingthe plasmid pMTOII4:

The plasmid pMTOII4 obtained in Example 9 was used to transform theEscherichia coli W3110 strA strain. An Ap^(r) colony obtained wascultured in the same manner as in Example 11, and cells were recoveredby centrifuging the culture at 7,000 rpm for 5 minutes. The cells weredissolved in the sample buffer of Laemmli et al., and the solution washeated and subjected to SDS-polyacrylamide gel electrophoresis after themethod of Laemmli et al., followed by Coomassie Brilliant Blue staining.As a result, a polypeptide band was detected at a position correspondingto a molecular weight of about 15,000. Since the pMTOII4-freeEscherichia coli W3110 strA strain gave no corresponding band, it wasevident that the pMTOII4-carrying transformant of Escherichia coli w3110strA had produced a Leu¹³ !motilin polymer protein as fused with a partof the salmon growth hormone.

EXAMPLE 14

Production of a Leu¹³ motilin polymer protein in Escherichia coli usingthe plasmid pMTOIII4:

The plasmid pMTOIII4 obtained in Example 10 was used to transform theEscherichia coli W3110 strA strain. An Ap^(r) colony obtained wascultured in the same manner as in Example 11, and cells were recoveredby centrifuging the culture at 7,000 rpm for 5 minutes. The cells weredissolved in the sample beffer of Laemmli et al., and the solution washeated and subjected to SDS-polyacrylamide gel electrophoresis after themethod of Laemmli et al., followed by Coomassie Brilliant Blue staining.As a result, a polypeptide band was detected at a position correspondingto a molecular weight of about 15,000. Since the pMTOIII4-freeEscherichia coli W3110 strA strain gave no corresponding band, it wasevident that the pMTOIII4-carrying transformant of Escherichia coliW3110 strA had produced a Leu¹³ !motilin polymer protein as fused with apart of the salmon growth hormone.

EXAMPLE 15

Production of a Leu¹³ motilin polymer protein in Escherichia coli usingplasmid pMTN4:

The plasmid pMTN4 obtained in Example 6 was used to transform theEscherichia coli W3110 strA strain. An Ap^(r) colony obtained wascultured into 8 ml of LG medium, followed by cultivation at 30° C. for 8hours. A part of the culture was inoculated into 10 ml of LG medium.After 16 hours of cultivation at 30° C., the culture was inoculated into1 liter of MCG medium supplemented with 100 μg/ml tryptophan and 50μg/ml ampicillin, and cultivation was carried out in a jar fermenter at30° C. for 48 hours.

A 100-ml portion of the culture was centrifuged at 7,000 rpm. The cellsthus recovered were washed with PSG (97 mM disodium phosphate, 1.5 mMpotassium dihydrogen phosphate, 137 mM NaCl, 2.7 mM KCl), suspended in60 ml of PBS and sonicated for 30 minutes. The sediment obtained bycentrifugation at 10,000 rpm for 40 minutes was dissolved in 3.48 ml of20 mM sodium phosphate buffer (pH 7.0), followed by addition of 3 ml ofdistilled water, 3.6 ml of 1.5M NaCl and 25 ml of Percol (Pharmacia FineChemicals) and centrifugation at 17,000 rpm for 15 minutes. The sedimentwas fractionated into two fractions according to the Percol densitygradient, and a higher-density fraction was recovered. To this fractionwas added 5 volumes of distilled water. Centrifugation at 11,000 rpm for7 minutes gave a sediment, which was washed with distilled water to give47 mg of granules. The protein guantitation was performed by using aprotein assay kit (Bio-Rad).

EXAMPLE 16

Production of Leu¹³ !motilin monomer:

The granular motilin polymer obtained in Example 15 (about 5 mg) wasdissolved in 2.0 ml of 70% formic acid, a solution of 42 mg of cyanogenbromide in 0.4 ml of 70% formic acid was added, and the mixture wasallowed to stand at 37° C. for 1 day. Again, 0.4 ml of a solution of 42mg of cyanogen bromide in 70% formic acid was added, and the wholemixture was allowed to stand overnight at 37° C. The product composed ofLeu¹³ !motilin monomer and the spacer peptide residue bound thereto(peptide 25) was isolated by high performance liquid chromatography(HPLC). An about 100-μg portion of the peptide isolated was dissolved in0.4 ml of 0.2M N-ethylmorpholine acetate buffer (pH 8.0), and thesolution was allowed to stand at 37° C. for 21 hours, whereby thehomoserine-derived lactone ring at the carboxyl terminus of peptide 25was cleaved. Then, 2 μg of carboxypeptidase A (Sigma) was added, and themixture was allowed to stand at 37° C. for 30 minutes to give peptide 26composed of Leu¹³ !motilin and arginine bound to the carboxyl terminusof the Leu¹³ !motilin. An about 22-μg portion of this peptide 26 wasdissolved in 0.2 ml of 0.2M N-ethylmorpholine acetate buffer (pH 8.0), 1μg of carboxypeptidase B (Sigma) was added, and the mixture was allowedto stand at 37° C. for 10 minutes. Thereafter, 0.2 ml of 0.1%trifluoroacetic acid solution was added to thereby terminate thereaction. In this way, Leu¹³ !motilin was obtained in a quantitativeyield. The structure of this Leu¹³ !motilin was confirmed by amino acidsequence analysis and mass analysis.

EXAMPLE 17

Intestine-contracting activity of Leu¹³ !motilin:

Male rabbits weighing 2.3-2.8 kg were sacrificed by exsanguination, andduodenum specimens, about 1.5 cm long, were excised. Each duodenumspecimen was suspended in a 30-ml Magnus bath, the lower end was tied toan isotonic transducer (Nihon Kohden model TD-112S), and the contractilerespond to the duodenum was recorded on a recorder (Yokogawa HokushinElectric type 3066). The duodenum was loaded with a tension of 1 g.

The experiment was carried out at a temperature of 28°±1° C. in a mixedgas atmosphere composed of 95% O₂ and 5% CO₂, using as the nutritivesolution Tyrode solution (8.0 g/l NaCl, 0.2 g/l KCl, 0.2 g/l CaCl₂, 0.1g/l MgCl₂, 0.05 g/l NaH₂ PO₄, 1.0 g/l NaHCO₃, 1.0 g/l glucose).

For evaluating the contracting activity of Met¹³ !motilin and that ofLeu¹³ !motilin, the contractile tensions obtained upon cumulativeaddition thereof to Tyrode solution to concentrations of 1×10⁻⁹ to3×10⁻⁷ g/ml were measured and the measured values were compared with thecontractile tension measured following addition of 10⁻⁵ g/ml ofacetylcholine and expressed in percentages with the value foracetylcholine being taken as 100%. The results obtained, which are shownin FIG. 17, show that Leu¹³ !motilin is comparable inintestine-contracting activity to naturally occurring porcine motilin.

REFERENCE EXAMPLE 1

Construction of the ATG vector pTrS20:

Following the scheme shown in FIG. 12, the ATG vector pTrS20 wasconstructed. In this vector, the SD sequence is 14 bases apart from theinitiation codon ATG, and the vector contains a SacI site immediatelybehind the ATG codon.

First, 3 μg of pKYP10 prepared by the method described in EuropeanPatent Publication No. 83069A was dissolved in 30 μl of Y-100 buffer, 6units each of the restriction enzymes BanIII and NruI (New EnglandBioLabs) were added, and the cleavage reaction was effected at 37° C.for 3 hours. From the reaction mixture, there was obtained, by the LGTmethod, about 0.5 μg of a Ptrp-containing DNA fragment of about 3.8 kb(BanIII-NruI fragment).

Separately, for providing the initiation codon ATG downstream from Ptrp,the following DNA linker was synthesized by the phosphotriester method:##STR4## The 19-mer and 17-met synthetic DNAs (10 picomoles each) weredissolved in a total volume of 20 μl of a solution containing 50 mMTris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM dithiothreitol, 0.1 mM EDTA and 1mM ATP, followed by addition of 3 units of T4 polynucleotide kinase(Takara Shuzo). The phosphorylation reaction was then carried out at 37°C. for 60 minutes.

Then, 0.1 μg of the above-mentioned pKYP10-derived BanIII-NruI fragment(about 3.8 kb) and about 0.5 picomole of the above-mentioned DNA linkerwere dissolved in 20 μl of T4 ligase buffer and, in addition, 2 units ofT4 DNA ligase was added. Then, the ligation reaction was conducted at 4°C. for 18 hours.

The thus-obtained recombinant plasmid mixture was used to transform theEscherichia coli HB101 strain (Boliver et al., Gene, 2, 75 (1977) ), andAp^(r) colonies were isolated. The plasmid DNA was recovered from thecultured cells derived from one of the colonies. The structure of theplasmid obtained was confirmed by agarose gel electrophoresis followingcleavage with the restriction enzymes EcoRI, BanIII, HindIII, SacI andNruI. This plasmid was named pTrS20. pTrS20 has the base sequence shownbelow in the neighborhood of the BanIII and HindIII sites was confirmedby the dideoxy sequencing method using M13 phage. ##STR5##

REFERENCE EXAMPLE 2

Construction of the salmon growth hormone expression plasmid psGHIM1(FIGS. 15 and 16):

In 40 μl of a solution (hereinafter, "Y-100 buffer") containing 20 mMTris-HCl (pH 7.5), 10 mM MgCl₂ and 100 mM NaCl, there was dissolved 3 μgof pGEL1 (about 3.4 kb), followed by addition of 5 units of BanIII(Toyobo). Then the digestion reaction was effected at 37° C. for 3hours. Extraction of the reaction mixture with phenol and precipitationwith ethanol gave about 2.4 mg of pGEL1, the product of cleavage at oneBanIII site. This DNA fragment (about 2.4 mg) was dissolved in 50 μl ofa solution (hereinafter, "DNA polymerase buffer") containing 50 mMTris-HCl (pH 7.8), 7 mM MgCl₂ and 6 mM mercaptoethanol, dATP and dTTPwere added each to a concentration of 1 mM, 5 units of DNA polymerase I(New England Bio-Labs) was further added, and the reaction was effectedat 37° C. for 30 minutes to thereby scrape off the projecting ends.About 2.0 μg of a DNA fragment was recovered by extraction with phenoland precipitation with ethanol. A 1-μg portion of said DNA fragment wasdissolved in 30 μl of a buffer (hereinafter, "T4 ligase buffer I")containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 10 mM dithiothreitoland 1 mM ATP, 2 units of T4 DNA ligase (Takara Shuzo: hereinafter thesame shall apply) was added, and the ligation reaction was conducted at4° C. for 8 hours. The reaction mixture was used to transform theEscherichia coli HB101 strain (Boliver et al., Gene, 2, 75 (1977)) bythe method of Cohen et al. (S. N. Cohen et al., Proc. Natl. Acad. Sci.USA, 69, 2110 (1972)), and Ap^(r) colonies were isolated. The plasmidDNA was separated from one of the transformant strains by the knownmethod (H. C. Birnboim et al., Nucleic Acids Res., 7, 1513 (1979)). Thuswas obtained pGEL10 (about 3.4 kb). The structure of pGEL10 wasconfirmed by agarose gel electrophoresis following cleavage with EcoRI,PstI, HindIII and BamHI. The base sequence from the SD sequencedownstream from the trp promoter to the translation initiation codon ATGfor the interferon-γ gene was determined by the Maxam-Gilbert method(Proc. Natl. Acad. Sci., USA, 74, 560 (1977)) and found to include 10 bpas follows: ##STR6##

The plasmid pGEL10 (5 μg) obtained above was dissolved in 40 μl of Y-100buffer, 10 units each of HindIII and BamHI were added, and the cleavagereaction was effected at 37° C. for 3 hours. From the reaction mixture,there was recovered about 2 μg of a DNA fragment (about 2.7 kb)containing the trp promoter region, origin of replication andlipoprotein terminator by the freezing-thawing method.

Separately, about 5 μg of psGHIB2 (about 3.8 kb) (prepared by the methodof Reference Example 3) was dissolved in 40 μl of Y-100 buffer, 10 unitsof BamHI were added, and the reaction was carried out at 37° C. for 3hours to complete cleavage. Then, i unit of HindIII was added, and thereaction was carried out at 37° C. for 30 minutes for partial cleavage.From the reaction mixture, there was recovered about 0.7 μg of a DNAfragment (about 1.1 kb) coding for the mature-form salmon growth hormoneby the freezing-thawing method.

About 0.1 μg of the DNA fragment of pGEL10 and about 0.2 μg of the DNAfragment of psGHIB2, both as recovered in the above manner, weredissolved in 30 μl of T4 DNA ligase buffer I, 2 units of T4 DNA ligasewas added, and the ligation reaction was effected at 4° C. for 18 hours.The reaction mixture was used to transform the Escherichia coli HB101strain, and the plasmid DNA was recovered from one of the coloniesobtained. Thus was obtained psGHIM1. The structure of psGHIM1 wasconfirmed by agarose gel electrophoresis following cleavage with EcoRI,HindIII, BamHI and PstI.

REFERENCE EXAMPLE 3

Construction of the recombinant plasmid psGHIB2 coding for themature-form salmon growth hormone:

A 5-μg of the plasmid psGH1 (prepared by the method described inEuropean Patent Publication No. 166444A) containing a DNA coding for thesalmon growth hormone was dissolved in 40 μl of a solution (hereinafter,"Y-10 buffer") containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 10mM NaCl, 10 units of the restriction enzyme MboII (New England BioLabs)was added, and the digestion reaction was effected at 37° C. for 3hours. Then, the NaCl concentration in the resulting solution wasadjusted to 175 mM, 10 units of SalI was added, and the digestionreaction was performed at 37° C. for 3 hours. From the reaction mixture,there was obtained, by the LGT method, about 0.2 μg of a 163 bp DNAfragment corresponding to the N terminus and its neighborhood.

Then, 5 μg of psGH1 was dissolved in 40 μl of Y-100 buffer, 10 units ofBamHI was added, and the digestion reaction was effected at 37° C. for 3hours. The NaCl concentration of the reaction mixture was then adjustedto 175 mM, 10 units of SalI was added, and the digestion reaction wascarried out at 37° C. for 3 hours. From the reaction mixture, there wasobtained, by the LTG method, about 0.5 μg of a DNA fragment (about 900bp) containing the C-terminal side and the 3'-nontranslational region.

Separately, 5 μg of pGEL1 was dissolved in 40 μl of Y-100 buffer, 10units each of BamHI and HindIII were added, and the digestion reactionwas carried out at 30° C. for 3 hours. From the reaction mixture, therewas obtained about 1 μg of a tryptophan promoter-containing DNA fragment(about 2.7 kb).

Further, separately, a DNA linker having the sequence given below wassynthesized for introducing the translation initiation codon requiredfor the expression of the DNA coding for the mature-form salmon growthhormone and for linking the vector DNA and the above DNA. ##STR7##

First, the single-stranded 17-mer and 12-mer DNAs were synthesized bythe conventional phosphotriester method (R. Crea et al., Proc. Natl.Acad. Sci., USA, 75, 5765 (1978)). The 17-mer and 12-mer single-strandedDNAs (12 picomoles each) were dissolved in 20 μl of a solutioncontaining 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM dithiothreitoland 1 mM ATP, 6 units of T4 polynucleotide kinase (Takara Shuzo) wereadded, and the phosphorylation reaction was carried out at 37° C. for 60minutes.

The psGH1-derived MboII-SalI fragment (163 bp) (0.1 picomole), 0.06picomole of the SalI-BamHI fragment (about 900 bp) and 0.02 picomole ofthe pGEL1-derived HindIII-BamHI fragment (about 2.7 kb), each obtainedas described above, were dissolved in 30 μl of a solution containing 50mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM dithiothreitol and 1 mM ATP. Tothe solution was added 5 μl of the phosphorylated syntheticDNA-containing reaction mixture mentioned above. To the resultantmixture was added 6 units of T4 ligase (Takara Shuzo), and the ligationreaction was effected at 4° C. for 18 hours.

The reaction mixture was used to transform the Escherichia coli HB101strain, Ap^(r) colonies were isolated, and the plasmid DNA was recoveredfrom one of the colonies. Thus was obtained psGHIB2 shown in FIG. 16.The structure of psGHIB2 was confirmed by agarose gel electrophoresisfollowing cleavage with EcoRI, HindIII, ClaI, BglII, SalI and BamHI. Thesequence in the neighborhood of the N terminus of the DNA coding for thesalmon growth hormone in psGHIB2 was determined by the Sanger's methodusing M13 phage and found to be as follows: ##STR8## As a result, it wasfound that psGHIB2 contains a DNA coding for the mature-form salmongrowth hormone polypeptide. A strain of Escherichia coli carrying theplasmid psGHIB2, namely Escherichia coli ESGHIB2, has been deposited asof Sep. 20, 1984 at the Fermentation Research Institute, Agency ofIndustrial Science and Technology under the deposit number FERM BP-612.

REFERENCE EXAMPLE 4

Construction of the plasmid pGHD7 (FIG. 13):

About 2 μg of the plasmid pGHB3 (European Patent Publication No.152613A; IGHB3, FERM BP-403) carrying the lecI promoter (of EuropeanPatent Publication No. 152613A), Escherichia coli lipoprotein gene (1pp) terminator and human interferon-γ cDNA was dissolved in 30 μl ofY-50 buffer (buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 7 mMMgCl₂ and 6 mM 2-mercaptoethanol), 8 units of PvuII were added, and thedigestion reaction was conducted at 37° C. for 2 hours.

Then, NaCl was added to make its concentration 150 mM, 8 units of SalIwere added, and the digestion reaction was further conducted at 37° C.for 2 hours. The DNA fragment obtained by extraction of the mixtureresulting from the digestion reaction with phenol and chloroform andprecipitation with ethanol was dissolved in a total volume of 30 μl of abuffer containing 50 mM Tris-HCl (pH 7.6), 7 mM MgCl₂, 6 mM2-mercaptoethanol, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP and 0.24 mMdTTP, 4 units of Escherichia coli-derived DNA polymerase I, Klenowfragment (Takara Shuzo) was added, and the reaction was carried out at15° C. for 2 hours to thereby convert the projecting ends resulting fromdigestion to blunt ends. After 10 minutes of heat treatment at 65° C.,the larger DNA fragment (3.6 kb) was purified by low-melting-pointagarose gel electrophoresis.

The thus-obtained DNA fragment (about 0.1 μg) was subjected to ligation.The ligation reaction was performed in 20 μl of a buffer containing 20mM Tris-HCl (pH.7.6), 10 mM MgCl₂, 10 mM dithiothreitol and 8.5 mM ATP(hereinafter, "T4 DNA ligase buffer II") in the presence of 2 units ofT4 DNA ligase at 4° C. for 18 hours.

The thus-obtained recombinant plasmid DNA was used to transform theEscherichia coli HB101 strain, and ampicillin-resistant strains wereisolated. The plasmid DNA was isolated from one of the transformantstrains and analyzed for its structure. As a results, it was confirmedthat the plasmid pGHD7 having the desired structure had beenconstructed.

REFERENCE EXAMPLE 5

Construction of the plasmid pArg4 (FIG. 14):

About 3 μg of the trp portable promoter-containing plasmid pKYP100 (T.Nishi et al., Agric. Biol. Chem, 48, 669-675 (1984)) was dissolved in 30μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 7 mMMgCl₂ and 6 mM 2-mercaptoethanol, 10 units of PstI and 10 units ofHindIII were added, and the digestion reaction was effected at 37° C.for 2 hours. After 10 minutes of heat treatment at 65° C., the smallerDNA fragment (0.88 kb) was purified by low-melting-point agarose gelelectrophoresis.

Furthermore, about 3 μg of the human interferon-γ expression plasmidpGEL1 (FERM BP-612; European Patent Publication No. 166444A) wasdissolved in 30 μl of Y-100 buffer, 10 units of PstI and 10 units ofNcoI were added, and the digestion reaction was conducted at 37° C. for2 hours. After 10 minutes of heat treatment at 65° C., the larger DNAfragment (1.7 kb) was purified by low-melting-point agarose gelelectrophoresis.

A DNA linker (having the EcoRV site and SalI site within itself) wasdesigned for use in coupling the two purified DNA fragments mentionedabove, as follows: SEQ ID NO:19 ##STR9##

Thus, two single-stranded DNAs (each 36-mer) shown above weresynthesized by the conventional phosphotriester method (R. Crea et al.,Proc. Natl. Acad. Sci., USA, 75, 5765 (1978)). Each DNA (20 picomoles)was dissolved in a total volume of 20 μl of a solution containing 50 mMTris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM dithiothreitol, 0.1 mM EDTA and 1mM ATP, 4 units of T4 polynucleotide kinase was added, and thephosphorylation reaction was conducted at 37° C. for 30 minutes. Equalamounts of these single-stranded DNAs were mixed, heated at 65° C. for 5minutes and then gradually cooled to room temperature, whereby a DNAlinker having the above structure was obtained.

This DNA linker (1 picomole) and the two above-mentioned purified DNAfragments (0.1 μg each) were ligated together in 20 μl of of theabove-mentioned T4 ligase buffer II in the presence of 2 units of T4 DNAligase at 4° C. The ligation reaction was performed for 18 hours.

The thus-obtained recombinant plasmid DNA was used to transform theEscherichia coli HB101 strain, and ampicillin-resistant strains wereobtained. The plasmid DNA was isolated from one of these transformantstrains and analyzed for its structure. It was confirmed that there hadbeen constructed he plasmid pArg4 having the desired structure.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /product=" LEU13!MOTILIN"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       PheValProIlePheThrTyrGlyGluLeuGlnArgLeuGlnGluLys                              151015                                                                        GluArgAsnLysGlyGln                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGATCAGATCTTCATGTTCGTTCCGATTTTCACTTACGGTGAACTGCAAC50                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGTTCACCGTAAGTGAAAATCGGAACGAACATGAAGATCTGAT43                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTCTGCAAGAGAAAGAACGTAACAAAGGTCAGCGGATCCTGTAAGAGCT49                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTTACAGGATCCGCTGACCTTTGTTACGTTCTTTCTCTTGCAGACGTTGC50                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGATCAGATCTTCATGTTCGTTCCGATTTTCACTTACGGTGAACTGCAACGTCTGCAAGA60                GAAAGAACGTAACAAAGGTCAGCGGATCCTGTAAGAGCT99                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTTACAGGATCCGCTGACCTTTGTTACGTTCTTTCTCTTGCAGACGTTGCAGTTCACCGT60                AAGTGAAAATCGGAACGAACATGAAGATCTGAT93                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCTTATGATAGAAAACCAACGGCTCTTCCA31                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCTGGAAGAGCCGTTGGTTTTCTATCATA31                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 26                                                              (D) OTHER INFORMATION: /note= "Xaa is homoserine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      PheValProIlePheThrTyrGlyGluLeuGlnArgLeuGlnGluLys                              151015                                                                        GluArgAsnLysGlyGlnArgIlePheXaa                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      PheValProIlePheThrTyrGlyGluLeuGlnArgLeuGlnGluLys                              151015                                                                        GluArgAsnLysGlyGlnArg                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CGATAAGCTTATGAGCTCG19                                                         (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGAGCTCATAAGCTTAT17                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note= "Shine-Dalgano sequence"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AAGGGTATCGATAAGCTTATGAGCTCGCGA30                                              (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAGGGTATAAGCTTATG17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AGCTTATGATAGAAAAC17                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TTTTCTATCATA12                                                                (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AAGCTTATGATAGAAAACCAA21                                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AGCTTATGATATCGAACGTCGACGACGGCGTCGAAC36                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CATGGTTCGACGCCGTCGTCGACGTTCGATATCATA36                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ArgIlePheMet                                                                  __________________________________________________________________________

What is claimed is:
 1. A method of producing a peptide having the aminoacid sequence set forth in SEQ ID NO:1 which method comprises the stepsof:(a) cultivating, in a nutrient growth medium, a microorganismharboring an expression vector which contains a recombinant DNA codingfor a peptide having said amino acid sequence inserted therein andallowing the peptide having said amino acid sequence to be expressed andto accumulate in the cultivated cells, and (b) harvesting said peptidefrom said cells.
 2. A method of producing a peptide having the aminoacid sequence set forth in SEQ ID NO:1 which comprises degrading apeptide having the amino acid sequence set forth in SEQ ID NO:10 withcarboxypeptidase A and carboxypeptidase B.